Creating E.coli constructs in yeast cells - If you are fed up with bacteria (Jun/29/2006 )

One of the biggest problem with ligation and stuff is its very bad reputation for creating large constructs.

Here is a general method that gaurantees 100% success ( I have used similar techniques since I work with yeast) to create your ordinary constructs. Unfortunately you have to be familiar with yeast transformation (which is extreemly easy) and and then rescue the plasmid to ecoli. The huge advantage is creating huge constructs is a piece of cake since you can join more than 2 fragments in one vector.

You can utilize the RecE/T system in e.coli for any DNA fragment so interest. It's flexible enough for homologous double or single recombination methods for insertions or replacements respectively. Its more commonly known as Red/ET recombination, but I haven't used it in a while and don't want to steer forum readers down the wrong path.

I don't, however, think that the rec system can accomadate multiple inerts in one cloning, but it may if the recombination sequences can vary for each integrating fragment...?

Some more well versed forum participants may be able to provide some good background papers.

-vasussci-

The problem with Red ET recombination is that it is not easy. You have to properly make your cells electrocompetent which is quite difficult. And then your competent must be more than 10^8 which is not that difficult. Moreover you cannot use right a way some strains like stbl2 or stbl4 that are great since they have to be transformed with certain plasmids and they are not easy to make electro competent

-shaq141-

QUOTE (shaq141 @ Jun 29 2006, 10:20 AM)

The problem with Red ET recombination is that it is not easy. You have to properly make your cells electrocompetent which is quite difficult. And then your competent must be more than 10^8 which is not that difficult. Moreover you cannot use right a way some strains like stbl2 or stbl4 that are great since they have to be transformed with certain plasmids and they are not easy to make electro competent

I really don't think either of these procedures are practical for high-throughput cloning.

What exactly would you be cloning that's over 20 kb? If you guys are really obsessed with this in vivo recombination stuff, look into gap repair with Acinetobacter calcoaceticus; it seems a lot easier than dealing with Yeast.